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Two distinct pathways of RNA polymerase backtracking determine the requirement for the Trigger Loop during RNA hydrolysis

Lookup NU author(s): Dr Hamed Mosaei Sejzi, Professor Nikolay Zenkin

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Abstract

© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Publication metadata

Author(s): Mosaei H, Zenkin N

Publication type: Article

Publication status: Published

Journal: Nucleic Acids Research

Year: 2021

Volume: 49

Issue: 15

Pages: 8777-8784

Print publication date: 07/09/2021

Online publication date: 07/08/2021

Acceptance date: 25/07/2021

Date deposited: 13/10/2021

ISSN (print): 0305-1048

ISSN (electronic): 1362-4962

Publisher: Oxford University Press

URL: https://doi.org/10.1093/nar/gkab675

DOI: 10.1093/nar/gkab675

PubMed id: 34365509


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